ABSTRACTAutophagy targets pathogens, damaged organelles and protein aggregates for lysosomal degradation. These ubiquitylated cargoes are recognized by specific autophagy receptors, which recruit LC3-positive membranes to form autophagosomes. Subsequently, autophagosomes fuse with endosomes and lysosomes, thus facilitating degradation of their content; however, the machinery that targets and mediates fusion of these organelles with autophagosomes remains to be established. Here we demonstrate that myosin VI, in concert with its adaptor proteins NDP52, optineurin, T6BP and Tom1, plays a crucial role in autophagy. We identify Tom1 as a myosin VI binding partner on endosomes, and demonstrate that loss of myosin VI and Tom1 reduces autophagosomal delivery of endocytic cargo and causes a block in autophagosome-lysosome fusion. We propose that myosin VI delivers endosomal membranes containing Tom1 to autophagosomes by docking to NDP52, T6BP and optineurin, thereby promoting autophagosome maturation and thus driving fusion with lysosomes.

Figure 7: Loss of Tom1 inhibits the maturation of autophagosomes and their subsequent fusion with lysosomes(a) Hela cells transiently transfected with siRNA against Tom1 were left untreated or treated with 100 nM Bafilomycin A1 for 4 hours. Western blot analysis was performed on whole cell lysates against the indicated proteins. (b) Quantitation of LC3-II intensity of Western blots were performed by an infrared imaging system. (+/− s.d.) (n=3) *p<0.05, **p<0.01 (c) Confocal immunofluorescence microscopy was performed on Hela cells transfected with Tom1 siRNA to evaluate LC3 punctae formation (green). Inserts provide higher magnification of boxed regions. Nuclei are labeled with Hoechst (blue). Scale bar, 20 μm. Quantitation of LC3-positive punctae (c) and p62-positive punctae (d) was performed in 96-well format on an Arrayscan VTi HCS microscope. Cells were identified by Hoechst staining of nuclei (blue). Results are represented as the average punctae fluorescence/cell (+/− s.d.) from n=3 independent experiments each performed in triplicate wells, each with >500 cells. (e) Hela cells with stable expression of the RFP-GFP-LC3 reporter construct were subjected to mock or Tom1 siRNA mediated knockdown. Confocal immunofluorescence microscopy was performed and images were subsequently quantitated for the correlation between the GFP and RFP signals using the ImageJ JACoP plugin. Results are of at least 100 individual cells from n=3 independent experiments and are represented as the Pearson’s coefficient of GFP/RFP overlap (+/− s.d.). A higher Pearson’s coefficient of GFP/RFP signal overlap represents a greater number of autophagosomes compared to autolysosomes. Scale bar, 20 μm. (f) Confocal immunofluorescence microscopy of Tom1 depleted RPE cells stably expressing GFP-LC3 immunostained against GFP (green) and Cathepsin D (red). Hoechst labels nuclei (blue). Insets represent magnified boxed regions. Scale bar, 20 μm. (g) Hela cells with stable expression of HttQ72-GFP were transiently transfected with Tom1 siRNA, saponin-extracted, and processed for immunofluorescence microscopy to quantitate HttQ72-GFP punctae. Immunolabelling against GFP (green) and p62 (red) was performed. Nuclei (blue) were labeled with Hoechst. Results are represented as the number of GFP-expressing cells with greater than 15 GFP-positive spots/cell (+/− s.d.) (n=3). Scale bar, 20 μm.

Mentions:
Next, we determined whether Tom1 is required for autophagy. SiRNA-mediated knockdown of Tom1 leads to an accumulation of LC3-II (Figure 7a,b; Supplementary Figure S8a), and an increase in the number of LC3-positive autophagosomes and p62-positive punctae visualised by immunofluorescence microscopy (Figure 7c,d; Supplementary Figure S8b), very similar to the phenotype observed in myosin VI knockdown cells (Figure 1a-d). In the Hela RFP-GFP-LC3 stable cell line, loss of Tom1 expression leads to an accumulation of LC3-positive, yellow autophagosomes that are Cathepsin D- and Lamp1-negative (Figure 7e,f; Supplementary Figure S2a). We also observed in Tom1 knockdown cells a defect in protein aggregate clearance and thus an increase in the number of cells with multiple p62 and LC3-positive HttQ72-GFP aggregates (Figure 7g; Supplementary Figure S3), phenocopying the knockdown of myosin VI (Figure 2d,e; Supplementary Figure S3). Given the very similar requirement for Tom1 and myosin VI during late stages of autophagy, we next tested whether Tom1 is recruited to autophagosomes and whether this localisation was myosin VI-dependent. As shown in figure 8a, endogenous Tom1 is present on LC3-positive autophagosomes. However, siRNA knockdown of myosin VI causes an increase in the number of Tom1-positive vesicles that are adjacent to, but not completely colocalising with LC3-positive autophagosomes, suggesting a defect in docking or fusion of these vesicles with autophagosomes (Figure 8a). In summary, myosin VI and Tom1 function together in the final stages of autophagy, since the loss of either protein leads to an accumulation of autophagosomes unable to mature to an autolysosome.

Figure 7: Loss of Tom1 inhibits the maturation of autophagosomes and their subsequent fusion with lysosomes(a) Hela cells transiently transfected with siRNA against Tom1 were left untreated or treated with 100 nM Bafilomycin A1 for 4 hours. Western blot analysis was performed on whole cell lysates against the indicated proteins. (b) Quantitation of LC3-II intensity of Western blots were performed by an infrared imaging system. (+/− s.d.) (n=3) *p<0.05, **p<0.01 (c) Confocal immunofluorescence microscopy was performed on Hela cells transfected with Tom1 siRNA to evaluate LC3 punctae formation (green). Inserts provide higher magnification of boxed regions. Nuclei are labeled with Hoechst (blue). Scale bar, 20 μm. Quantitation of LC3-positive punctae (c) and p62-positive punctae (d) was performed in 96-well format on an Arrayscan VTi HCS microscope. Cells were identified by Hoechst staining of nuclei (blue). Results are represented as the average punctae fluorescence/cell (+/− s.d.) from n=3 independent experiments each performed in triplicate wells, each with >500 cells. (e) Hela cells with stable expression of the RFP-GFP-LC3 reporter construct were subjected to mock or Tom1 siRNA mediated knockdown. Confocal immunofluorescence microscopy was performed and images were subsequently quantitated for the correlation between the GFP and RFP signals using the ImageJ JACoP plugin. Results are of at least 100 individual cells from n=3 independent experiments and are represented as the Pearson’s coefficient of GFP/RFP overlap (+/− s.d.). A higher Pearson’s coefficient of GFP/RFP signal overlap represents a greater number of autophagosomes compared to autolysosomes. Scale bar, 20 μm. (f) Confocal immunofluorescence microscopy of Tom1 depleted RPE cells stably expressing GFP-LC3 immunostained against GFP (green) and Cathepsin D (red). Hoechst labels nuclei (blue). Insets represent magnified boxed regions. Scale bar, 20 μm. (g) Hela cells with stable expression of HttQ72-GFP were transiently transfected with Tom1 siRNA, saponin-extracted, and processed for immunofluorescence microscopy to quantitate HttQ72-GFP punctae. Immunolabelling against GFP (green) and p62 (red) was performed. Nuclei (blue) were labeled with Hoechst. Results are represented as the number of GFP-expressing cells with greater than 15 GFP-positive spots/cell (+/− s.d.) (n=3). Scale bar, 20 μm.

Mentions:
Next, we determined whether Tom1 is required for autophagy. SiRNA-mediated knockdown of Tom1 leads to an accumulation of LC3-II (Figure 7a,b; Supplementary Figure S8a), and an increase in the number of LC3-positive autophagosomes and p62-positive punctae visualised by immunofluorescence microscopy (Figure 7c,d; Supplementary Figure S8b), very similar to the phenotype observed in myosin VI knockdown cells (Figure 1a-d). In the Hela RFP-GFP-LC3 stable cell line, loss of Tom1 expression leads to an accumulation of LC3-positive, yellow autophagosomes that are Cathepsin D- and Lamp1-negative (Figure 7e,f; Supplementary Figure S2a). We also observed in Tom1 knockdown cells a defect in protein aggregate clearance and thus an increase in the number of cells with multiple p62 and LC3-positive HttQ72-GFP aggregates (Figure 7g; Supplementary Figure S3), phenocopying the knockdown of myosin VI (Figure 2d,e; Supplementary Figure S3). Given the very similar requirement for Tom1 and myosin VI during late stages of autophagy, we next tested whether Tom1 is recruited to autophagosomes and whether this localisation was myosin VI-dependent. As shown in figure 8a, endogenous Tom1 is present on LC3-positive autophagosomes. However, siRNA knockdown of myosin VI causes an increase in the number of Tom1-positive vesicles that are adjacent to, but not completely colocalising with LC3-positive autophagosomes, suggesting a defect in docking or fusion of these vesicles with autophagosomes (Figure 8a). In summary, myosin VI and Tom1 function together in the final stages of autophagy, since the loss of either protein leads to an accumulation of autophagosomes unable to mature to an autolysosome.

Bottom Line:
Here we demonstrate that myosin VI, in concert with its adaptor proteins NDP52, optineurin, T6BP and Tom1, plays a crucial role in autophagy.We identify Tom1 as a myosin VI binding partner on endosomes, and demonstrate that loss of myosin VI and Tom1 reduces autophagosomal delivery of endocytic cargo and causes a block in autophagosome-lysosome fusion.We propose that myosin VI delivers endosomal membranes containing Tom1 to autophagosomes by docking to NDP52, T6BP and optineurin, thereby promoting autophagosome maturation and thus driving fusion with lysosomes.

ABSTRACTAutophagy targets pathogens, damaged organelles and protein aggregates for lysosomal degradation. These ubiquitylated cargoes are recognized by specific autophagy receptors, which recruit LC3-positive membranes to form autophagosomes. Subsequently, autophagosomes fuse with endosomes and lysosomes, thus facilitating degradation of their content; however, the machinery that targets and mediates fusion of these organelles with autophagosomes remains to be established. Here we demonstrate that myosin VI, in concert with its adaptor proteins NDP52, optineurin, T6BP and Tom1, plays a crucial role in autophagy. We identify Tom1 as a myosin VI binding partner on endosomes, and demonstrate that loss of myosin VI and Tom1 reduces autophagosomal delivery of endocytic cargo and causes a block in autophagosome-lysosome fusion. We propose that myosin VI delivers endosomal membranes containing Tom1 to autophagosomes by docking to NDP52, T6BP and optineurin, thereby promoting autophagosome maturation and thus driving fusion with lysosomes.